Methods and compositions that increase pesticidal activity for fr901228

ABSTRACT

The present invention includes methods and compositions that produce greater pesticidal activity in FR901228 conjugates. FR901228 can be conjugated with one or more thiol containing molecules and produce a new conjugate that exhibits increase pesticidal activity than FR901228 alone.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/123,580 filed on Dec. 10, 2020 and is incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to the field of pesticides.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is described in connection with pesticides. More specifically, methods and compositions that increase pesticidal activity of FR901228.

FR901228 is a known molecule that can be isolated from microbes such as Chromobacterium sp., or more particularly, Chromobacterium violaceum WB968 strain (FERM BP-1968) in nutrient medium and has been found to be an antibacterial agent and/or antitumor agent (see, for example, U.S. Pat. No. 7,396,665, which is incorporated herein in its entirety). In addition, FR901228 can also be isolated from Burkholderia sp. such as Burkholderia rinojensis A396 and is known to be pesticidal (see, for example, U.S. Pat. No. 9,701,673 B2, which is incorporated herein in its entirety).

In addition to the fermentation method mentioned above, it is known that FR901228 can also be prepared by semi-synthesis or whole synthesis utilizing techniques known in the art (J. Am. Chem. Soc., 118, 7237-7238 (1996)).

Although FR901228 is known to be pesticidal, there is a need to further improve its pesticidal activity. As such, the present disclosure relates to methods and compositions that increases the pesticidal activity of FR901228.

SUMMARY OF THE INVENTION

In an aspect, the present invention relates to methods for increasing pesticidal activity of FR901228 comprising conjugating the FR901228 with one or more thiol molecule, wherein said thiol molecule comprises at least one thiol functional group in its chemical structure, and wherein the FR901228 and the one or more thiol molecule are linked via at least one disulfide bond, and wherein said conjugated molecule exhibits increased pesticidal activity as compared to FR901228 alone.

In an embodiment, said pesticidal activity mentioned for the above method includes virucidal, herbicidal, germicidal, fungicidal, nematicidal, bactericidal and/or insecticidal activity.

In an aspect, said one or more thiol molecule is a protein and/or a peptide. Non-limiting examples include protein HtpG and/or DnaK. In an embodiment, the protein HtpG and/or DnaK are from Burkholderia A396 NRRL Accession No. B-50319.

Yet in another aspect, said FR901228 is derived or isolated from one or more microbe or E. coli. In another embodiment, the FR901228 is chemically synthesized.

In an aspect, said microbe comprises Chromobacterium sp. and/or Burkholderia sp. Non-liming examples include Chromobacterium violaceum WB968 strain. Burkholderia A396 NRRL Accession No. B-50319.

In one embodiment, the present invention relates to a pesticidal combination comprising FR901228 and one or more thiol molecule, wherein said FR901228 and said one or more thiol molecule are chemically linked through one or more disulfide bond, and wherein said combination exhibits increased pesticidal activity as compared to FR901228 alone.

In an embodiment, said pesticidal activity for the above-mentioned combination includes virucidal, herbicidal, germicidal, fungicidal, nematicidal, bactericidal and/or insecticidal activity.

In an aspect, the pesticidal combination mentioned above, wherein said one or more thiol molecule is a protein and/or a peptide. Non-limiting examples include protein HtpG and/or

DnaK. In one embodiment, the protein HtpG and/or DnaK are from Burkholderia A396 NRRL Accession No. B-50319.

Yet in another aspect, the pesticidal combination mentioned above, wherein said FR901228 is chemically synthesized.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

FIG. 1 denotes the chemical structure of FR901228.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

As used herein, the term “pesticidal” or “pesticidal activity” includes, but are not limited to: virucidal, herbicidal, germicidal, fungicidal, nematicidal, bactericidal and/or insecticidal activity.

As used herein, the term “conjugating” or “conjugated” refers to physically attaching two or more molecules together via chemical bonds. The method of attaching could be via traditional chemical synthesis routes or simply by placing two molecules together in a suitable environment (e.g., by in close proximity and/or by supplying heat) where the attachment can take place. A non-limiting example of conjugating is via disulfide bonds when both molecules have available thiol functional group(s) present in their chemical structure that can be linked.

As used herein, FR901228 is also known as romidepsin and has the chemical structure as denotes in FIG. 1. In the present disclosure, the terms “FR901228”, “MW 540” and “romidepsin” are used interchangeably as they refer to the same molecule.

The term “peptide” as used in this disclosure refers amino acid sequences that are typically shorter in nature, as compared to a protein. Both protein and peptide are made up of amino acids, and the present disclosure merely denotes their typical difference in length as understood by one skilled in the art.

In one embodiment, the present disclosure relates to methods and compositions that increases pesticidal activity of FR901228. MW 540 contains a disulfide bond capable of interacting with thiol residues of cysteine-containing proteins (Sevier and Kaiser 2002) or molecules (large or small), forming more disulfide bonds. These disulfide bond-scrambling reactions are prompted, for example, by heat. The present inventors isolated MW 540 and conjugated MW 540 with one or more thiol molecules that contain one or more thiol functional group via the formation of disulfide bond(s). The present inventors found that this newly formed conjugated molecule has higher pesticidal activity as compare to MW 540 alone. For example, the insecticidal activity can be increased as compared to MW 540 alone.

In one embodiment, MW 540 can conjugate with any thiol molecule(s) that contain one or more thiol function groups. MW 540 itself has a disulfide bond, and the sulfur atom(s) can interact with another molecule's thiol group and form one or more disulfide bonds. It is known in the art that MW 540 can interact with itself to form dimer, trimer... etc. since each molecule can interact with itself via disulfide bonds.

A non-limiting example of another molecule with thiol groups is a protein having cystine residues. A non-limiting example of a protein having cystine residues is chaperone protein HtpG from Burkholderia rinojensis A396 having the following amino acid sequence:

(SEQ ID No.: 1) MTQQTMSFQAEVKQLLHLMIHSLYSNKEIFLRELVSNASDAADKLRFEAL ENGALYENDPNLRIRIGFDPAARTLTIDDNGIGMSRDEAIANLGTIARSG TKEFFSKLSGDQQKDAALIGQFGVGFYSGFIVADRITVETRRAGLPASEG VRWESGGEGDFSIDAIERAARGTTITLHLREGEDELLSAHRLKSIVRKYS DHVALPILMQQEAWDAEKGEMVAKDEDETVNQASALWTRAKSEITDEQYQ QFYQHLAHDHQNPLAWTHNRVEGRSEYTQLLYVPSHAPFDLWNRDYRGGL KLYVKRVFIMDDAEQLLPQYLRFVKGVVDSADLPLNVSREILQESRDVKA IREGVTKRALSMLEELANAEDDAGKEQYRTFWSAFGQVLKEGVGEDQANR ERVAKLVRFASTHGGTDAQDVSLADYVARMKPEQTRIYYVTADTWQAATH SPHLEVFRKKGVEVLLLTDRVDEWMLSYLQEFDGKPLASVARGDLDLGAL DDAEKKAQEETGEAFKPLVEKMKEALGDKAKDVRVTFRLTDSPSCLVADD HDMSGYLQRMLKAAGQSGPAMQPILEVNPEHPLVKQLQADSPEFGDWCHL LFDQALLAEGGALEDPASFVKRTNTLLLSRAA.

Another non-limiting example of a protein having cystine residues is chaperone protein DnaK from Burkholderia rinojensis A396 having the following amino acid sequence:

(SEQ ID No.: 2) MGKIIGIDLGTTNSCVAVMEGNQVKVIENSEGARTTPSIIAYMDDNEVLV GAPAKRQSVTNPRNTLFAVKRLIGRRFEEKEVQKDIGLMPYAIIKADNGD AWVEAHGDKLAPPQVSAEVLRKMKKTAEDYLGEPVTEAVITVPAYFNDSQ RQATKDAGRIAGLEVKRIINEPTAAALAFGLDKAEKGDRKIAVYDLGGGT FDVSIIEIADVDGEMQFEVLSTNGDTFLGGEDFDQRIIDYIIGEFKKEQG VDLSKDVLALQRLKEAAEKAKIELSSGQQTEINLPYITADASGPKHLNLK ITRAKLEALVEELVERTIEPCRIAIKDAGVKVSDIDDVILVGGQTRMPKV LEKVKEFFGKDPRRDVNPDEAVAVGAAIQGQVLSGDRKDVLLLDVTPLSL GIETLGGVMTKMINKNTTIPTKHAQVYSTADDNQGAVTIKVFQGEREMAA GNKLLGEFNLEGIPPAPRGVPQIEVTFDIDANGILHVGAKDKATGKENKI TIKANSGLSEAEIDQMIKDAEANAAEDHKLRELADSRNQGDALVHSTKKA VAEYGDKLDAGEKDKIEAALKELEDVLKNTSADKAAIDAKIEALSTASQK LGEKMYADMQAQQAGAAGAAGAAEGAAHGGAQQADDVVDADFKEVKKD.

Additional molecules having thiol functional groups are contemplated by this disclosure and can conjugate with 1\4W 540. Non-limiting example include GroES and GroEL heat shock chaperone proteins.

In one embodiment, the thiol containing molecule can be chemically synthesized or derived from microbes and are in the free form. In another embodiment, the thiol containing molecule can reside on a microbe's cell wall. MW 540 can be added to the above-mentioned cell wall to increase the pesticidal activity of MW 540.

Examples

Example 1

In bacteria such as E. coli, chaperonins (proteins that aid the assembly and folding of other protein molecules in living cells) are produced during heat exposure (often described as heat-shocking). Cytosolic chaperonins, including HtpG and/or DnaK, were evaluated in this example. The proteins can be overexpressed in E. coli as a method of production. These proteins contain 2 cysteines that have reactive thiol sidechains and can bind to MW 540.

The present inventors evaluated whether these two proteins can form conjugates with MW 540 and whether the conjugate has the same insecticidal activity as MW 540 alone. One skilled in the art knows that protein purification often uses polyhistidine-tags to assist with the concentration and removal of desired proteins from cellular material. The two protein sequences were cloned into an expression vector (pET26b(+)) and over-expressed in E. coli BL21 cells with a His tag added to the C-terminus of the sequence. In other words, His-tagged HtpG and DnaK from B. rinojensis A396 were overexpressed in E. coli and were purified by immobilized metal ion affinity chromatography. Since E. coli does not produce MW540 or have insecticidal activity against Spodoptera exigua, it is a suitable surrogate for these experiments.

After mixing MW 540 with HtpG and/or DnaK, heat was applied to the mixture to from disulfide bond conjugates and the end-product was evaluated and its insecticidal activity determined. Quantification of MW 540 and derivatives before and after heating in the presence of recombinant HtpG and DnaK are shown in Table 1 below. Note that MW 540 is capable of forming a dimer or trimer with another MW 540 via disulfide bonds. Therefore, the dimers are designated as MW1080, and the trimer is designated as MW 1620 . . . etc.

TABLE 1 Before heating After heating MW540/ MW540 MW540/ MW540 Free MW540 1080/1620 conjugate Free MW540 1080/1620 conjugate HtpG + MW540 31 36.9 Not detected 9.7 20.4 17.0 DnaK + MW540 37 44.1 Not detected 16 25.3 19.5

Table 1 shows that the conjugates were formed. Table 1's numbers are expressed as protein concentrations as μg/mL. Subsequently, the insecticidal activity of the end products was evaluated using Beet army worm diet overlay assay known in the art. See, for example, U.S. Patent No. 9,701,673 B2.

As shown in Table 2 and Table 3 below, both end products of HtpG and DnaK plus MW 540 conjugates have increased insecticidal activity as compared to MW 540 alone. Control experiments were also performed, where Table 4 shows that the HtPG and DnaK alone does not exhibit insecticidal activity by itself.

TABLE 2 MW 540 Protein LC₅₀ Concentration Concentration (μg/mL Sample (μg/mL) (mg/mL) MW 540) MW 540 50 0 0.27 HtpG + MW 540 50 0.537 0.12 HtpG + MW 540 50 0.0537 0.27 HtpG + MW 540 50 0.00537 0.26

TABLE 3 MW 540 Protein LC₅₀ Concentration Concentration (μg/mL Sample (μg/mL) (mg/mL) MW 540) MW 540 50 0 0.35 DnaK + MW 540 50 1.40 0.11 HtpG + MW 540 50 0.940 0.14 HtpG + DnaK + MW 540 50 2.34 0.08

TABLE 4 MW 540 Protein LC₅₀ Concentration Concentration (μg/mL Sample (μg/mL) (mg/mL) MW 540) MW 540, unheated 50 0 0.43 HtpG + MW 540, heated 50 5 0.14 DnaK + MW 540, heated 50 5 0.23 HtpG, heated 0 5 No Activity DnaK, heated 0 5 No Activity HtpG and DnaK, unheated 0 10 No Activity

Combining both HtpG and DnaK with MW 540 resulted in the highest activity. In addition, HtpG and DnaK did not show insecticidal activity on their own regardless of heat treatment.

These results indicate at least the following:

An insecticidal conjugate can be formed between MW 540 and a protein or protein-like molecule having one or more thiol functional groups.

The protein type is not specific to B. rinojensis A396.

It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. 

What is claimed is:
 1. A method for increasing pesticidal activity of FR901228 comprising: conjugating the FR901228 with one or more thiol molecule, wherein said thiol molecule comprises at least one thiol functional group in its chemical structure, and wherein the FR901228 and the one or more thiol molecule are linked via at least one disulfide bond, and wherein said conjugated molecule exhibits increased pesticidal activity as compared to FR901228 alone.
 2. The method of claim 1, wherein said pesticidal activity comprises insecticidal activity.
 3. The method of claim 1, wherein said one or more thiol molecule is a protein and/or a peptide.
 4. The method of claim 3, wherein said protein comprises HtpG and/or DnaK.
 5. The method of claim 1, wherein said FR901228 is derived or isolated from one or more microbe or E. coli.
 6. The method of claim 5, wherein said microbe comprise Chromobacterium sp. or Burkholderia sp.
 7. The method of claim 6, wherein said Chromobacterium sp. is Chromobacterium violaceum WB968 strain.
 8. The method of claim 6, wherein said Burkholderia sp. is Burkholderia A396 NRRL Accession No. B-50319.
 9. The method of claim 1, wherein said FR901228 is chemically synthesized.
 10. A pesticidal combination comprising FR901228 and one or more thiol molecule, wherein said FR901228 and said one or more thiol molecule are chemically linked through one or more disulfide bond, and wherein said combination exhibits increased pesticidal activity as compared to FR901228 alone.
 11. The pesticidal combination of claim 10, wherein said pesticidal activity comprises insecticidal activity.
 12. The pesticidal combination of claim 10, wherein said one or more thiol molecule is a protein and/or a peptide.
 13. The pesticidal combination of claim 12, wherein said protein comprises HtpG and/or DnaK.
 14. The pesticidal combination of claim 10, wherein said FR901228 is chemically synthesized. 